Cloning and functional characterization of the SUR2/SYR2 gene encoding sphinganine hydroxylase in Pichia ciferrii

Abstract

Saccharomyces cerevisiae sphinganine C4-hydroxylase encoded by the SUR2 gene catalyses the conversion of sphinganine to phytosphingosine. We isolated the SUR2 gene from Pichia ciferrii using nucleotide sequence homology to S. cerevisiae SUR2 to study hydroxylation of sphinganine in the sphingoid base overproducing yeast P. ciferrii. A positive clone was confirmed by nucleotide sequencing. A syringomycin-E resistance phenotype of a S. cerevisiae sur2-null mutant was complemented by expression of the cloned P. ciferrii SUR2 gene. Restoration of phytosphingosine production in the complemented strain was also confirmed, indicating that the cloned gene is a functional homologue of S. cerevisiae SUR2. The nucleotide sequence of the P. ciferrii SUR2 gene was submitted to GenBank under Accession No. AY151276.

basA Regulates Cell Wall Organization and Asexual/Sexual Sporulation Ratio in Aspergillus nidulans

“…The functional similarity of Sur2 homologs in a broad range of species such as S. cerevisiae (Haak et al 1997), P. ciferrii (Bae et al 2004), A. nidulans (this study), and even in plants, as described in A. thaliana (Sperling et al 2001), strongly suggest that this protein group has a conserved enzymatic activity in various eukaryotic systems.…”

“…2 (Grilley et al 1998). Genes encoding Sur2 homologs in Pichia ciferrii (Bae et al 2004) and in Arabidopsis thaliana (Sperling et al 2001) are also required for DHS C4 hydroxylation, suggesting that the biochemical activity of Sur2 homologs is highly conserved in a wide range of species. Other than yeast, the physiological role of Sur2 homologs has not been investigated in other fungi.…”

Metabolic engineering of the non-conventional yeast Pichia ciferrii for production of rare sphingoid bases

“…The isolation of P. ciferrii homologs of MTS1 encoding sphingolipid C-9-methyltransferase and HSX11 encoding glucosylceramide synthase was not attempted. It should be noted that the gene encoding the P. ciferrii sphingolipid C-4-hydroxylase has meanwhile been identified independently by another group (Bae et al, 2004).…”

An Efficient Genome-Wide Fusion Partner Screening System for Secretion of Recombinant Proteins in Yeast

“…The haploid yeasts, S. cerevisiae Y2805 ( Mat α pep4::HIS3 prb1 can1 his3-200 ura3-52 ) and Y2805 Δgal80 (Y2805 gal80::Tc190 ) 47 48 49 were used as general hosts for gene expression and genomic or cDNA isolation. The invertase-defective strain Y2805 Δgal80Δsuc2 (Y2805 gal80::Tc190 suc2::Tc190 ) were constructed by targeted gene disruption with the URA3 pop-out cassette 50 . All yeast transformations were performed using the lithium acetate method 51 .…”